Name: how equilibrium binding constant affects binding
Uploaded: 2023-04-30T18:28:41.000Z
Duration: 2 min 18 s
Channel: JoVE

In der Zelle kollidieren Proteine zufällig mit anderen Molekülen, wie z. B. anderen Proteinen, Nukleinsäuren und niedermolekularen Liganden.
Wenn die Bindung unspezifisch ist, führen einige nicht-kovalente Wechselwirkungen zwischen den Molekülen zu einer kurzen Assoziation.
Bindet ein bestimmter Ligand an das Protein, bildet er ausgedehnte nicht-kovalente Wechselwirkungen entlang komplementärer Oberflächen. Solche Komplexe sind stabil und bleiben lange Zeit gebunden, bevor sie sich dissoziieren.
Die Stärke einer Bindungswechselwirkung wird in Bezug auf ihre Gleichgewichtskonstante Kb angegeben, die auch als Bindungs- oder Assoziationskonstante bezeichnet wird. 
Kb kann aus dem Verhältnis der Konzentration des Protein-Liganden-Komplexes zu den Konzentrationen des ungebundenen Proteins und des Liganden im Gleichgewicht berechnet werden. 
Aufgrund seiner Beziehung zur Änderung der freien Energie aufgrund der Bindung bedeutet ein großes Kb eine starke Abnahme von Delta G, was auf eine starke Affinität zwischen Protein und Liganden hinweist.
Für die Protein-Liganden-Bindung sind zwei konkurrierende Prozesse wichtig: die Assoziation eines Proteins und eines Liganden zu einem Komplex und die Dissoziation des Komplexes in die Reaktanten.
Die Assoziationskonstante kon ist ein Maß für die Anzahl der Bindungsereignisse pro Sekunde zwischen einem Protein und seinem Liganden; sie kann verwendet werden, um die Bindungsrate des Liganden an das Protein bei einer bestimmten Konzentration zu berechnen. 
Umgekehrt ist koff ein Maß für die Anzahl der Dissoziationsereignisse; es kann verwendet werden, um zu berechnen, wie schnell der Komplex auseinanderfällt.
Wenn die Assoziationsrate der Dissoziationsrate entspricht, wird ein Gleichgewicht erreicht, bei dem die Nettokonzentrationen von Produkten und Reaktanten konstant bleiben.
Somit ist im Gleichgewicht kon mal das Produkt aus den Gleichgewichtskonzentrationen des Proteins und des Liganden gleich koff mal der Gleichgewichtskonzentration des Protein-Liganden-Komplexes.
Die Neuanordnung dieses Ausdrucks zeigt auch, dass das Verhältnis von kon zu koff gleich Kb im Gleichgewicht ist.

<ol>
	<li>Pollard, Thomas D. "Ein Leitfaden für einfache und informative Bindungsassays." Molekularbiologie der Zelle 21, Nr. 23 (2010): 4061-4067.</li>
	<li>Hulme, Edward C. und Mike A. Trevethick. "Ligandenbindungsassays im Gleichgewicht: Validierung und Interpretation." Britisches Journal für Pharmakologie 161, Nr. 6 (2010): 1219-1237.</li>
	<li>Medina-Franco, José L., Oscar Méndez-Lucio und Karina Martinez-Mayorga. "Das Zusammenspiel zwischen molekularer Modellierung und Chemoinformatik zur Charakterisierung von Protein-Liganden- und Protein-Protein-Interaktionslandschaften für die Wirkstoffforschung." In Fortschritte in der Proteinchemie und Strukturbiologie, Bd. 96, S. 1-37. Akademischer Verlag, 2014.</li>
</ol>

die gleichgewichtsbindungskonstante und die bindungsstärke

The Equilibrium Binding Constant and Binding Strength

Die Gleichgewichtsbindungskonstante (Kb) quantifiziert die Stärke einer Protein-Ligand-Wechselwirkung. Kb kann wie folgt berechnet werden, wenn sich die Reaktion im Gleichgewicht befindet:

<img alt="Eq1" src="/files/ftp_upload/11513/Equation 1.png" style="text-align: center; width:650px;" />

wobei P und L das ungebundene Protein bzw. der ungebundene Ligand sind und PL der Protein-Ligand-Komplex ist.

Da die Menge des gebundenen Liganden auch mit der Geschwindigkeit der Ligandenbindung zusammenhängt, können Experimente auch Kb bestimmen, indem die Geschwindigkeiten der Protein-Ligand-Assoziation (kon) und der Dissoziation (koff) unter Verwendung des folgenden Verhältnisses untersucht werden:

<img alt="Eq1" data-cke-saved-src="/files/ftp_upload/11513/Equation 2.png" src="/files/ftp_upload/11513/Equation 2.png" style="text-align: center;width:650px;" "="">

Daher werden zwei Kategorien von Bindungstests verwendet, um die Gleichgewichtsbindungskonstante zu bestimmen – solche, die Gleichgewichtskonzentrationen messen, und solche, die die Kinetik einer Reaktion messen. In diesem Fall muss sich die Reaktion zum Zeitpunkt der Messung im Gleichgewicht befinden.

Die Methode zur Bestimmung der Gleichgewichtskonzentrationen hängt von der gewünschten Empfindlichkeit und der Einfachheit der Signalerkennung ab. Aus diesen Gründen werden spektroskopische Tests am häufigsten verwendet. Bei diesen Experimenten führt die Reaktion zu einer Absorptionsänderung eines Reaktanten oder Produkts bei einer bestimmten Wellenlänge, die mit einem UV-Vis-Spektrophotometer nachweisbar ist. Alternativ kann der Reaktant oder das Produkt mit einer fluoreszierenden Sonde markiert werden oder einen intrinsischen Fluorophor enthalten. Anschließend kann der Reaktionsfortschritt anhand der Fluoreszenzänderung gemessen werden. Diese Tests werden durchgeführt, indem die Konzentrationen eines Reaktanten variiert werden, während der Rest des Experiments konstant gehalten wird. Die Ergebnisse können dann grafisch dargestellt und mit verschiedenen Kurvenanpassungsmethoden analysiert werden.

Wechselwirkungen zwischen Proteinen und Liganden werden auch mithilfe verschiedener biochemischer und spektroskopischer Techniken untersucht. Die Strukturanalyse mittels Röntgenkristallographie und NMR-Spektroskopie hilft bei der Vorhersage von Protein-Ligand-Wechselwirkungen durch molekulare Simulationen. Theoretische und rechnerische Ansätze, wie beispielsweise Protein-Ligand-Docking-Studien, werden in großem Umfang verwendet, um die Position und Wechselwirkungen kleiner Molekülliganden, einschließlich Arzneimittelkandidaten, zu charakterisieren. Computergestütztes Arzneimitteldesign ist eine schnelle und kostengünstige Alternative, um das Tempo herkömmlicher Trial-and-Error-Arzneimitteltests zu beschleunigen.

Die Gleichgewichtsbindungskonstante und die Bindungsstärke

Die Gleichgewichtsbindungskonstante (Kb) quantifiziert die Stärke einer Protein-Liganden-Wechselwirkung. Kb kann wie folgt berechnet werden, wenn die Reaktion bei ...

the-equilibrium-binding-constant-and-binding-strength

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how equilibrium binding constant affects binding

The equilibrium binding constant describes how strongly two molecules bind at equilibrium. It is a useful way to compare binding strength in molecular interactions.

This topic focuses on how binding is measured and what the equilibrium binding constant means. A higher value indicates stronger binding, while a lower value indicates weaker binding. Understanding this constant helps explain how tightly molecules stay together under equilibrium conditions.

The page also connects the binding constant to real binding strength. It shows why this value matters when comparing different molecular pairs. For high school students, it provides a clear link between a chemistry concept and molecular behavior.

How Equilibrium Binding Constant Affects Binding

how-equilibrium-binding-constant-affects-binding

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the equilibrium binding constant and binding strength

The equilibrium binding constant describes how strongly two molecules bind at equilibrium. It is a useful way to compare binding strength in molecular interactions. This topic focuses on how binding is measured and what the equilibrium binding constant means. A higher value indicates stronger binding, while a lower value indicates weaker binding. Understanding this constant helps explain how tightly molecules stay together under equilibrium conditions. The page also connects the binding...

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key terms and concepts

Learn how the equilibrium binding constant reflects binding strength and why higher or lower values matter in molecular interactions at equilibrium.

How Equilibrium Binding Constant Affects Binding | JoVE K12

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How Equilibrium Binding Constant Affects Binding

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